The long term goal of the applicant's research is to improve the efficiency and the safety of gene delivery, which are the critical parameters for the clinical success of gene therapy. Based on the current knowledge on the barriers of nonviral gene delivery, the mechanisms of viral infection and the role of lysosome disruption in apoptosis, it is hypothesized that cationic lipids which can quickly hydrolyze into endosome-destabilizing fragments in response to a small drop of pH can improve both the efficiency and the safety of gene transfection mediated by their lipoplexes. Based on this hypothesis, two novel types of pH-sensitive cationic lipids, both containing an acid-labile ortho ester linker group are proposed. At neutral pH, these cationic lipids should be reasonably stable and be able to condense with plasmid DNA into cationic lipoplexes. Upon endocytosis, these lipids will be hydrolyzed into membrane-destabilizing fragments in response to the drop of pH in the endosomes. This will trigger the change of physicochemical properties of the lipoplexes, which will in turn destabilize the endosome and deliver the plasmid DNA into the cytoplasm. The project has four specific aims: 1) to design and prepare ortho ester-based cationic lipids and lipoplexes; 2) to study the pH-triggered hydrolysis of the ortho ester cationic lipids and the pH-triggered change of the physicochemical properties of their lipoplexes; 3) to evaluate the gene transfection efficiency (in cell culture and in mice) of the ortho ester-based cationic lipoplexes in relationship to their pH-sensitivity; 4) to evaluate the cytotoxicity of the ortho ester-based cationic lipoplexes in relationship to their pH-sensitivity, especially cytotoxicity caused by disruption of lysosome enzymes into the cytoplasm. In the short run, this project will discover pH sensitive cationic lipoplexes with improved gene transfection efficiency and safety. In the long run, the findings of this project can be used to develop optimized lipoplexes for gene delivery to local tissues or sterically stabilized lipoplexes for remote targeting to tumors. [unreadable] [unreadable]